{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T21:26:07Z","timestamp":1768512367920,"version":"3.49.0"},"posted":{"date-parts":[[2024,8,8]]},"group-title":"Microbiology","reference-count":35,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,8,8]],"date-time":"2024-08-08T00:00:00Z","timestamp":1723075200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,8,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Objective<\/jats:title>\n                  <jats:p>\n                    The fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that\n                    <jats:italic>hacA<\/jats:italic>\n                    is essential for\n                    <jats:italic>Aspergillus fumigatus<\/jats:italic>\n                    virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both\n                    <jats:italic>in vitro<\/jats:italic>\n                    and in a treatment model of FK.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    The antifungal activity of Ire1 inhibitors was tested against conidia of several\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    isolates by a microbroth dilution assay and against fungal biofilm by XTT reduction. The influence of 4\u03bc8C on\n                    <jats:italic>hacA<\/jats:italic>\n                    mRNA splicing in\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4\u03bc8C in the cornea was assessed by applying drops to uninfected or\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    -infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    Among six Ire1 inhibitors screened, the endonuclease inhibitor 4\u03bc8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    Af293 in the same concentration range that blocked\n                    <jats:italic>hacA<\/jats:italic>\n                    splicing and UPR gene induction (60-120 \u00b5M). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4\u03bc8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and &gt;90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>\n                    The\n                    <jats:italic>in vitro<\/jats:italic>\n                    data suggest that 4\u03bc8C displays antifungal activity against\n                    <jats:italic>A. fumigatus<\/jats:italic>\n                    through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2024.08.08.607189","type":"posted-content","created":{"date-parts":[[2024,8,8]],"date-time":"2024-08-08T23:55:11Z","timestamp":1723161311000},"source":"Crossref","is-referenced-by-count":1,"title":["The mammalian Ire1 inhibitor, 4\u00b58C, exhibits broad anti-\n                  <i>Aspergillus<\/i>\n                  activity\n                  <i>in vitro<\/i>\n                  and in a treatment model of fungal keratitis"],"prefix":"10.64898","author":[{"given":"Manali M.","family":"Kamath","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emily M.","family":"Adams","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jorge D.","family":"Lightfoot","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Becca L.","family":"Wells","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin K.","family":"Fuller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024081200501532000_2024.08.08.607189v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.eye.6700557"},{"key":"2024081200501532000_2024.08.08.607189v1.2","doi-asserted-by":"publisher","DOI":"10.1111\/ceo.14113"},{"key":"2024081200501532000_2024.08.08.607189v1.3","doi-asserted-by":"crossref","unstructured":"Atta S , Perera C , Kowalski RP , Jhanji V . 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